1. Field of the Invention
The present application relates generally to monitoring and altering the activity of cellular signaling pathways involving the protein Sam68.
2. Description of the Related Art
Sam68 is also known as KHDRBS1 (KH domain containing, RNA binding, signal-transduction associated 1). Sam68, a specific target of the Src tyrosine kinase in mitosis, is a KH domain containing RNA binding protein that also contains multiple proline rich regions that bind to SH2, SH3 and WW domain containing proteins (Ellis et al., 1990. Nature 343, 377-381) (Taylor and Shalloway, 1994. Nature 368, 867-871) (Lukong and Richard, 2003. Biochimica et biophysica acta 1653, 73-86). Sam68 also contains an RNA binding domain, the GRP33/SAM68/GLD-1 (GSG) domain (Reviewed in Bielli et al., 2011, Endocrine-Related Cancer 18: R91-R102). However, most of Sam68's function has been attributed to its RNA binding property by which it regulates metabolism, nuclear export and stability of RNA. In line with its functional diversity, Sam68 exists both in the cytoplasm as well as the nucleus and undergoes various post-translational modification including phosphorylation, methylation and acetylation, which modulates its function (Lukong and Richard, 2003. Biochimica et biophysica acta 1653, 73-86.). No known roles for Sam68 in cytokine receptor signaling have been reported before.
TNFα is a potent cytokine that plays an important role in inflammatory response, immunity, cell growth and differentiation as well as apoptosis. These biological processes are mediated by TNF binding to the TNF receptor 1 and TNF receptor 2, where the latter acts as a modulator of the signaling through the former in many cell types. The majority of the known functions of TNF occur through TNF receptor 1 triggering, which initiates either a pro-survival pathway, mainly through the activation of the transcription factor NF-κB, or an extrinsic apoptotic pathway through the activation of caspases under conditions where NF-κB activation or de novo protein synthesis is inhibited. In addition, TNF signaling also triggers MAP kinases activating JNK, p38 and ERK pathways, which also contribute to biological effects TNF (Locksley et al., 2001. Cell 104, 487-501) (Wallach et al., 1999. Annual review of immunology 17, 331-367) (Wajant et al., 2003. Cell death and differentiation 10, 45-65).
The NF-κB family of transcription factors is composed of five members: p65 (RelA), RelB, c-Rel, NF-κB1 (which occurs both as a precursor, p105, and in a processed form, p50), and NF-κB2 (which occurs both as a precursor, p100, and as its processed product, p52). Three of them, c-REL, RelB and RelA, have a carboxy-terminal non-homologous transactivation domain that activate transcription from NF-κB binding sites in target genes. In the resting state, NF-κB proteins exist as homo- or hetero-dimers in the cytoplasm bound to the inhibitory proteins of the IκB family. Activation occurs by signal induced phosphorylation, ubiquitylation and proteasomal degradation of the IκB proteins releasing the bound NF-κB proteins, which in turn translocates to the nucleus to initiate transcription (Ghosh et al., 1998). IκB's are phosphorylated by the upstream kinase complex called signalosome, mainly composed of IKK1, IKK2 and NEMO, which are activated following their recruitment to the TNFR (Hayden and Ghosh, 2008. Cell 132, 344-362.) (Vallabhapurapu and Karin, 2009. Annual review of immunology 27, 693-733.)
Apoptosis plays a role in maintenance of tissue homeostasis and development, supporting equilibrium between cellular proliferation and death. While the survival pathway mainly relies on activation of NF-κB (Baker and Reddy, 1998), apoptosis depends on activation of caspases (Varfolomeev et al., 1998. Immunity 9, 267-276.) (Wang et al., 2008. Cell 133, 693-703.). Apoptosis can occur either by an extrinsic pathway or by an intrinsic pathway. In the intrinsic pathway, non-receptor cellular stress like DNA damage or oxidative stress causes increase in mitochondrial permeability, which leads to release of pro-apoptotic factors in to the cytosol that activate caspases (Brenner and Mak, 2009. Current opinion in cell biology 21, 871-877.). In the extrinsic pathway, the apoptosis is initiated by a death inducing ligand binding to its cognate receptor in the membrane followed by recruitment, aggregation and activation of caspases (Park et al., 2007. Annual review of immunology 25, 561-586.). Signaling through TNF can induce apoptosis via the extrinsic pathway. Moreover, Fas is a prototypic death receptor that induce apoptosis by the extrinsic pathway in a TNF-independent manner (Krammer, 2000. Nature 407, 789-795.) (Wallach et al., 1999. Annual review of immunology 17, 331-367.).